Rho GTPases function as binary switches, cycling from GDP-bound "off" states to GTP-bound "on" states capable of engaging myriad effectors to coordinate a plethora of cellular responses that typically require changes in the cytoskeleton. For example, cellular responses that require Rho GTPases include: chemotaxis, differentiation and division, wound-healing, phagocytosis, axonal pathfinding, and polarized secretion. The cycling of guanine nucleotides bound to Rho GTPases is tightly controlled at several points. In particular, guanine nucleotide exchange factors (GEFs) directly bind Rho GTPases to accelerate the ejection of bound GDP and the loading of GTP. With approximately seventy members in humans, the Dbl-family of proteins represents the largest groups of GEFs for Rho GTPases. All Dbl-family members possess a characteristic Dbl homology (DH) domain necessary to engage Rho GTPases and catalyze exchange. A pleckstrin homology (PH) domain is found immediately C-terminal to the vast majority of DH domains. The nature of these associated PH domains is not fully understood, but in general, this tandem linkage serves to coordinate Rho GTPase activation with the binding of specific phosphoinositides to the DH-associated PH domains. Given the importance of Dbl-family GEFs in regulating the activation of Rho GTPases and the central role of these GTPases to numerous facets of cell biology, we are heavily invested in understanding the regulation of these exchange factors and their associated roles in controlling biological events. Accordingly, this application has three specific aims: 1. To define conserved mechanisms regulating the auto-inhibition of Dbl-family GEFs. 2. To define roles for a subset of Dbl-family GEFs in cancer and neuronal development. 3. To define general mechanisms underlying the cooperative functioning of DH/PH cassettes. Public Health Relevance: The dysregulated activation of Rho GTPases, often mediated by Dbl-family guanine nucleotide exchange factors (GEFs), is associated with numerous developmental, immunological, and proliferative diseases, including cancer. The proposed work is designed to illuminate conserved mechanisms regulating the activation of Rho GTPases by Dbl-family GEFs and to place this regulation within the context of models of cancer progression and neuronal development.